Secretory proteins produced in polarized epithelial cells are targeted to either the basolateral or apical domains of the plasma membrane by either constitutive or regulated pathways, or both. The intracellular routing of a secretory protein is most likely to be dependent on interaction(s) between components of the secretory apparatus and the secretory protein itself. It is unknown what signal(s) is (are) required to accomplish this. The objective of this research is to develop a system in which it will be possible to examine these signals in order to further knowledge about the various intracellular routes taken by secretory proteins, and, hopefully, the means by which those pathways are controlled. It is essential to develop such understanding of the secretory process since not only do all normal cells secrete physiologically relevant molecules, but many diseased cells exhibit impaired secretion. Two polarized epithelial cell lines will be used to map intracellular protein traffic, MDCK cells, possessing only the constitutive pathway, and bovine uterine epithelial (BUE) cells, whose apical pathway is thought to be regulated. It will first be confirmed that BUE cells do possess a regulated pathway. Immunofluorescence of intact and permeabilized BUE and MDCK cells have shown that laminin, a basement membrane protein, is secreted basolaterally and constitutively. Since no apically secreted proteins from these cells have been described, we will introduce plasmids containing the genes for ovalbumin (Ov), human growth hormone (hGH). and rat pancreatic ribonuclease (rpRNase) which have been coupled to promoters which allow them to be expressed. Synthesis will also be monitored by immunoprecipitation, SDS-PAGE, and autoradiography after labeling cells with (35S) methionine and/or by use of a solid-phase radioimmunoassay. Double labeling techniques utilizing protein A-gold electron microscopic immunocytochemistry will be employed to compare the intracellular distributions of laminin, Ov, hGH, or rpRNase in MDCK and BUE cells. The exogenous genes are secreted via regulated pathways in their cells of origin. Determination of how and where each is sorted in a foreign cell, i.e., via either a constitutive or regulated pathway, and to either the basolateral or apical surface or both, will furnish information on how protein traffic is controlled.